1. Field of the Invention
The present invention relates to a transgenic C. elegans which expresses an amyloid precursor protein (APP) or a part thereof, to the transgene itself, to the protein encoded by the transgene, and also to a process for preparing the transgenic C. elegans and to its use.
2. Description of Related Art
Several publications are referenced in the application. These references describe the state of the art to which this invention pertains, and are incorporated herein by reference.
Alzheimer""s disease (morbus Alzheimer) is a neurodegenerative disorder of the brain which, at the cellular level, is accompanied by a massive loss of neurons in the limbic system and in the cerebral cortex. At the molecular level, it is possible to detect protein depositions, so-called plaques, in the affected areas of the brain, which depositions constitute an important feature of Alzheimer""s disease. The protein which most frequently occurs in these plaques is a peptide of from 40 to 42 amino acids in size which is termed the Axcex2 peptide. This peptide is a cleavage product of a substantially larger protein of from 695 to 751 amino acids, the so-called amyloid precursor protein (APP).
APP is an integral transmembrane protein which traverses the lipid double layer once. By far the largest part of the protein is located extracellularly, while the shorter C-terminal domain is directed into the cytosol (FIG. 1). The Axcex2 peptide is shown in dark gray in FIG. 1. About two thirds of the Axcex2 peptide are derived from the extracellular domain of APP and about one third from the transmembrane domain.
In addition to the APP which is located in the membrane, it is also possible to detect a secreted form of the amyloid precursor protein, which form comprises the large ectodomain of the APP and is termed APPsec (xe2x80x9csecreted APPxe2x80x9d). APPsec is formed from APP by proteolytic cleavage which is effected by xcex1-secretase. The proteolytic cleavage takes place at a site in the amino acid sequence of APP which lies within the amino acid sequence of the Axcex2 peptide (after amino acid residue 16 of the Axcex2 peptide). Proteolysis of APP by the xcex1-secretase consequently rules out the possibility of the Axcex2 peptide being formed.
The Axcex2 peptide can consequently only be formed from APP by an alternative processing route. It is postulated that two further proteases are involved in this processing route, with one of the proteases, which is termed xcex2-secretase, cutting the APP at the N terminus of the Axcex2 peptide and the second protease, which is termed xcex3-secretase, releasing the C terminus of the Axcex2 peptide (Kang, J. et al., Nature, 325, 733) (FIG. 1).
It has not as yet been possible to identify any of the three secretases or proteases (xcex1-secretase, xcex2-secretase and xcex3-secretase). However, knowledge of the secretases is of great interest, in particular within the context of investigations with regard to Alzheimer""s disease and with regard to identifying the proteins involved, which proteins can then in turn be employed as targets in follow-up studies since, on the one hand, inhibition of the xcex2-secretase, and in particular of the xcex3-secretase, could lead to a decrease in Axcex2 production and, on the other hand, activation of the xcex1-secretase would increase the processing of APP into APPsec and thereby simultaneously reduce formation of the Axcex2 peptide.
There is a large amount of evidence that the Axcex2 peptide is a crucial factor in the development of Alzheimer""s disease. Inter alia, Axcex2 fibrils are postulated to be neurotoxic in cell culture (Yankner, B. A. et al., (1990) Proc Natl Acad Sci USA,87, 9020). Furthermore, the neuropathology which is characteristic of Alzheimer""s disease already appears at the age of 30 in Down""s syndrome patients, who have an additional copy of APP. In this case, it is assumed that overexpression of APP is followed by an increased conversion into the Axcex2 peptide (Rumble, B. et al., (1989), N. Engl. J. Med., 320,1446).
The familial forms of Alzheimer""s disease constitute what is probably the most powerful evidence of the central role of the Axcex2 peptide. In these forms, there are mutations in the APP gene around the region of the xcex2-secretase and xcex3-secretase cleavage sites or in two further AD-associated genes (presenilins) which, in cell culture, lead to a substantial increase in Axcex2 production (Scheuner, D. et al., (1996), Nature Medicine, 2, 864).
While C. elegans has already been used as a model organism in Alzheimer""s disease, these studies do not relate to the processing of APP into the Axcex2 peptide. Some of the studies are concerned with two other Alzheimer-associated proteins, i.e. the presenilins. The presenilins are transmembrane proteins which traverse the membrane 6-8 times. They are of great importance in familial cases of Alzheimers since specific mutations in the presenilin genes lead to Alzheimer""s disease. In this connection, it was shown that homologs to the human presenilins (sel-12, spe-4 and hop-1) are present in C. elegans, with the function of the presenilins being conserved in humans and worm (Levitan D, Greenwald I (1995) Nature 377, 351; Levitan et al.(1996) Proc Natl Acad Sci USA, 93, 14940; Baumeister R (1997) Genes and Function 1, 149; Xiajun Li and Iva Greenwald (1997) Proc Natl Acad Sci USA, 94, 12204).
Other studies deal with the APP homolog in C. elegans, which is termed Apl-1, and with expression of the Axcex2 peptide in C. elegans. However, Apl-1 does not possess any region which is homologous with the amino acid sequence of the Axcex2 peptide; C. elegans does not therefore possess any endogenous Axcex2 peptide (Daigle I, Li C (1993) Proc Natl Acad Sci USA, 90 (24), 12045).
C. D. Link, Proc Natl Acad Sci USA (1995) 92, 9368 described the expression of Axcex2 peptide (but not that of an Axcex2 precursor protein) in C. elegans. These studies involve preparing transgenic worms which express an Axcex21-42 peptide (i.e. the Axcex2 peptide which consists of 42 amino acids) as a fusion protein together with a synthetic signal peptide and under the control of the muscle-specific promoter unc 54. Muscle-specific protein depositions which reacted with anti-xcex2-amyloid antibodies were detected in the studies.
Other studies (e.g. C. Link et al. personal communication) relate to investigations of the aggregation and toxicity of the Axcex2 peptide in the C. elegans model system.
Transgenic C. elegans lines were established in the present study in order to investigate the existence of a processing machinery in C. elegans which is involved in the formation of Axcex2 peptide and to identify potential secretases in this worm.
In this invention, APP genes have been transferred into C. elegans to create a transgenic C. elegans organism. This transgenic C. elegans can then be used to investigate the processing machinery involved in the formation of the Axcex2 peptide and to identify potential secretases.
The present invention relates to a transgene (a gene that has been transferred from one species to another by genetic engineering) which contains
a) a nucleotide sequence encoding an amyloid precursor protein (APP) or a part thereof, wherein the nucleotide sequence comprising the APP peptide or part thereof, contains, as part of the sequence, a nucleotide sequence comprising a complete Axcex2 peptide or a part of the Axcex2 peptide, and
b) where appropriate, one or more further coding and/or non-coding nucleotide sequences, and
c) a promoter for expression in a cell of the nematode Caenorhabditis elegans (C. elegans).
The nucleotide sequence preferably encodes the 100 carboxyterminal amino acids of APP, beginning with the sequence of the Axcex2 peptide and ending with the carboxyterminal amino acid of APP (C100 fragment). The APP is preferably one of the isoforms APP695 (695 amino acids), APP751 (751 amino acids), APP770 (770 amino acids) and L-APP. All the isoforms are formed from the same APP gene by means of alternative splicing. In APP695, exons 7 and 8 were removed by splicing, whereas only exon 8 is lacking in APP751 and exon 7 and 8 are present in APP770. In addition to this, other splicing forms of APP exist in which exon 15 has been removed by splicing. These forms are termed L-APP and are likewise present in the forms which are spliced with regard to exons 7 and 8.
In one particular embodiment of the invention, the transgene contains the nucleotide sequence SEQ ID NO.: 1 or a part thereof or a sequence homologous to SEQ ID No. 1.
The transgene can preferably contain an additional coding nucleotide sequence which is located at the 5xe2x80x2 end of the nucleotide sequence encoding APP or a part thereof. In one particular embodiment of the invention, the additional nucleotide sequence encodes a signal peptide or a part thereof, for example encodes the APP signal peptide (SP) having the amino acid sequence SEQ ID NO.:9 or a part thereof. The sequence from the N terminus of the Axcex2 peptide to the C terminus of APP consists of 99 amino acids. The APP signal peptide consists of 17 amino acids. When a fusion product comprising the N terminus of the Axcex2 peptide to the C terminus of APP and the APP signal peptide is cloned, one or more spacer amino acids is/are preferably inserted between these two parts of the fusion product, with preference being given to inserting one amino acid, for example leucine. The C-terminal fragment is therefore given different designations, e.g. C100 (C=C terminus), LC99 (L=leucine), LC1-99, C99 or SPA4CT (SP=signal peptide, A4=Axcex2 peptide and CT=C terminus).
In one particular embodiment of the invention, the transgene contains the nucleotide sequence SEQ ID NO.: 2 or a part thereof and/or the nucleotide sequence SEQ ID NO.: 3 or a part thereof.
In addition to this, the transgene can also contain one or more additional non-coding and/or one or more additional coding nucleotide sequences.
For example, the transgene can contain, as an additional non-coding nucleotide sequence, a sequence from an intron of the APP gene, e.g. a sequence which is derived from the 42 bp intron of the APP gene and exhibits the sequence SEQ ID NO.: 4. A transgene which contains the nucleotide sequence SEQ ID NO.: 5 is part of the subject-matter of the invention.
The transgene also preferably contains one or more gene-regulating sequences for regulating expression of the encoded protein, preferably a constitutive promoter or a promoter which can be regulated. For example, the promoter can be active in the neuronal, muscular or dermal tissue of C. elegans or be ubiquitously active in C. elegans. A promoter can, for example, be selected from the group of the C. elegans promoters unc-54, hsp 16-2, unc-119, goa-1 and sel-12. In one particular embodiment of the invention, the transgene contains a promoter having the nucleotide sequence SEQ ID NO.: 6. In one particular embodiment, the transgene contains the nucleotide sequence SEQ ID NO.: 7.
The transgene can be present in a vector, for example in an expression vector. For example, a recombinant expression vector can contain the nucleotide sequence SEQ ID NO.: 8.
The invention also relates to the preparation of an expression vector, with a transgene being integrated into a vector in accordance with known methods. In particular, the invention relates to the use of an expression vector for preparing a transgenic cell, with it being possible for this cell to be part of a non-human organism, e.g. C. elegans. 
The invention also relates to the preparation of the transgene, with suitable part sequences being ligated in the appropriate order and in the correct reading frame, where appropriate while inserting linkers. In particular, the invention relates to the use of the transgene, for example for preparing a transgenic cell, with it being possible for this cell to be part of a non-human organism. For example, the cell can be a C. elegans cell.
One particular embodiment of the invention relates to a transgenic C. elegans which contains the transgene. The transgene can also be present in the C. elegans in an expression vector. The transgene can be present in the C. elegans intrachromosomally and/or extrachromosomally. One or more transgenes or expression vectors which contain the transgene can be present intrachromosomally and/or extrachromosomally as long tandem arrays. A transgenic cell or a transgenic organism preferably contains another expression vector as well, which vector contains a nucleotide sequence which encodes a marker, with the marker either being a temperature-sensitive marker or a phenotypic marker. For example, the marker can be a visual marker or a behaviorally phenotypic marker. Examples are fluorescent markers, e.g. GFP (green fluorescent protein) or EGFP (enhanced green fluorescent protein), marker genes which encode a dominant, mutated form of a particular protein, e.g. a dominant Rol6 mutation, or marker sequences which encode antisense RNA, e.g. the antisense RNA of Unc-22.
One or more copies of the transgene and/or of the expression vector and, where appropriate, of an additional expression vector are preferably present in the germ cells and/or the somatic cells of the transgenic C. elegans. 
The invention also relates to a process for preparing a transgenic C. elegans, with a transgene and/or an expression vector, where appropriate in the presence of an additional expression vector which contains a nucleotide sequence which encodes a marker, being microinjected into the germ cells of a C. elegans. A DNA construct which expresses SP-C100 (SP=signal peptide) under the control of a neuron-specific promoter can, for example, be used for preparing the transgenic C. elegans lines (FIG. 2). Since C100 is composed of the Axcex2 sequence and the C terminus of APP, only the xcex3-secretase cleavage is required in order to release the Axcex2 peptide from C100. C100 is also a substrate for the xcex3-secretase.
The invention also relates to the use of a transgenic C. elegans, for example for expressing an SP-C100 fusion protein. An SP-C100 fusion protein having the amino acid sequence SEQ ID NO.: 10 is part of the subject-matter of the invention.
In particular, the invention relates to the use of a transgenic C. elegans for identifying a xcex3-secretase activity and/or an xcex1-secretase activity in C. elegans, to its use in methods for identifying and/or characterizing substances which inhibit the xcex3-secretase activity, to its use in methods for identifying and/or characterizing substances which increase the xcex1-secretase activity, and to its use in methods for identifying and/or characterizing substances which can be used as active compounds for treating and/or preventing Alzheimer""s disease.
In the present study, the nematode Caenorhabditis elegans (C. elegans) was chosen as the model organism for identifying secretases which are involved in processing APP into the Axcex2 peptide. This worm is outstandingly suitable for genetic studies and has therefore in the past been employed on many occasions for investigating universally important processes such as programmed cell death, neuronal guidance and RAS/MAP kinase signaling (Riddle, D. L. et al. (1997)).
The important points which make C. elegans especially appropriate for such studies include the following (C. Kenyon, Science (1988) 240, 1448; P. E. Kuwabara (1997), TIG, 13, 454):
Its small genome, which is composed of about 19,000 genes or 97 Mb and which was sequenced completely in December 1998. (The C. elegans Sequencing Consortium, Science (1998), 282, 2012).
Its reproduction by self fertilization. In the case of the two sexes of C. elegans, a distinction is made between males and hermaphrodites, i.e. hermaphroditic animals which fertilize their eggs themselves before laying. A crucial advantage of this type of reproduction is that, after a transgene has been introduced into the germ line, a hermaphrodite can automatically generate homozygous transgenic descendants. There is therefore no need for any further crossing steps, as in the case of Drosophila, for example, for preparing transgenic lines.
Its easy handling in the laboratory due to its small size (about 1 mm in length) and its relatively undemanding growth conditions. As a result, a large number of worms can be handled routinely in the laboratory.
Its short generation time of 3 days, which makes it possible to obtain large quantities of biological material for analysis within a very short time.
A complete cell description for the development and anatomy of C. elegans is available.
Detailed genetic maps and methods for genetic analysis in C. elegans are available.
Technologies for preparing knock-out animals are available. In the same way, technologies exist for mutagenizing the C. elegans genome (transposon mutagenesis and ethyl methanesulfonate (EMS) mutagenesis).
The following are possible uses of the transgenic C. elegans lines:
1. Identification of a xcex3-secretase-like activity in C. elegans using mutagenesis approaches. It is planned that a transposon mutagenesis, which destroys the xcex3-secretase-like activity, should be carried out and that the corresponding gene should be sought by detecting the worms which no longer possess this activity. Such a screening method is described in the literature: Korswagen H. C. et al., (1996), 93, 14680 Proc Natl Acad Sci USA.
Alternative approaches would be mutagenesis using ethyl methanesulfonate (EMS) or else anti-sense RNA approaches. In the latter case, an attempt could be made to find motifs which were common to all C. elegans proteases and to downregulate these proteases specifically using anti-sense RNAs which were directed against these motifs. Screening for the Axcex2 peptide could then show whether one of the proteases was involved in Axcex2 peptide production.
2. Identification of a xcex3-secretase-like activity in C. elegans, perhaps by a similar route to that described in item 1.
3. Armed with knowledge of a xcex3-secretase or xcex3-secretase-like activity in C. elegans, it is possible to search for human xcex3-secretase or xcex3-secretase-like activity by means of a homology comparison.
4. Identification of drugs which
inhibit the activity of xcex3-secretase, in order to inhibit Axcex2 production from the amyloid precursor protein directly.
activate xcex3-secretase and thereby indirectly inhibit formation of the Axcex2 peptide by increasing APPsec production.
This approach could take place in a 96-well format since C. elegans can be maintained in suspension in 96-well plates.
Since the screening is carried out on a whole organism, it is possible, to a large extent, to exclude drugs which have an unspecific toxic effect.
5. Investigation of the aggregation behavior, and of a possible neurotoxic effect, of the Axcex2 peptide in C. elegans. Screening for drugs which inhibit aggregation of the Axcex2 peptide.
6. Investigation of the modulation of APP processing by other proteins (e.g. presenilins or ApoE) as a result of their overexpression or knock-out. Since the presenilins are Alzheimer-associated proteins and ApoE constitutes a risk factor in Alzheimer""s disease, these proteins could have an effect on formation of the Axcex2 peptide and, as a consequence, their role in the APP processing pathway could be investigated.
7. Where appropriate, validation of an xcex1-secretase and/or xcex3-secretase activity which has been found using other experimental approaches known to the skilled person.